Munishwar N. Gupta

Freeze‐drying of proteins: some emerging concerns

Freeze‐drying (lyophilization) removes water from a frozen sample by sublimation and desorption. It can be viewed as a three‐step process consisting of freezing, primary drying and secondary drying. While cryoprotectants can protect the protein from denaturation during early stages, lyoprotectants are needed to prevent protein inactivation during drying. The structural changes as a result of freeze‐drying have been investigated, especially by FTIR (Fourier‐transform IR) spectroscopy. In general, drying results in a decrease of α‐helix and random structure and an increase in β‐sheet structure. In the case of basic fibroblast growth factor and γ‐interferon, enhanced FTIR showed large conformational changes and aggregation during freeze‐drying, which could be prevented by using sucrose as a lyoprotectant. It is now well established that structural changes during freeze‐drying are responsible for low activity of freeze‐dried powders in nearly anhydrous media. Strategies such as salt activation can give ‘activated’ enzyme powders, e.g. salt‐activated thermolysin‐catalysed regioselective acylation of taxol to give a more soluble derivative for therapeutic use. In the presence of moisture, freeze‐dried proteins can undergo disulphide interchange and other reactions which lead to inactivation. Such molecular changes during storage have been described for human insulin, tetanus toxoid and interleukin‐2. Some successful preventive strategies in these cases have also been mentioned as illustrations. Finally, it is emphasized that freeze‐drying is not an innocuous process and needs to be understood and used carefully.

Lactose hydrolysis by Lactozym™ immobilized on cellulose beads in batch and fluidized bed modes

LactozymTM is a commercially available preparation of b-galactosida se from Kluyveromyces fragilis. It has valid generally recognized as safe (GRAS) status for whey hydrolysis and production of low lactose milk. Immobilized b-galactosidase from K. fragilis has been less studied. In this work, LactozymTM was immobilized on cellulose beads via epichlorohydrin coupling chemistry. The optimized preparation was characterized in terms of its kinetic parameters. The fluidized bed hydrolyzed whey lactose ( > 90% conversion) in 5 h as compared to 48 h taken by the same enzyme in continuous batch mode. The immobilized enzyme could be reused three times without any change in the performance of the fluidized bed reactor. The fluidized bed could also hydrolyze milk lactose up to 60% within 5 h. The above data show that this enzyme from a GRAS status source can also be used to develop a process for lactose hydrolysis for whey utilization as well as production of low lactose milk.

Polarity Index: The Guiding Solvent Parameter for Enzyme Stability in Aqueous-Organic Cosolvent Mixtures

Enzyme catalysis in aqueous–organic cosolvent mixtures has wide applications. However, inadequate attention has been paid to the issue of stability of enzymes in such media. The results with polyphenol oxidase, peroxidase, acid phosphatase, and trypsin show that solvents with polarity indexes of 5.8 and above are “good” solvents. These solvents when used as cosolvents in aqueous–organic solvent media do not denature the enzymes irreversibly. Enzyme(s) exposed to these solvents retain most of their activity even after 48 h of exposure, whereas solvents with polarity indexes of <5.1 denature the enzyme completely within 0–4 h in most of the cases studied. It appears that at higher concentrations (50% and above) cosolvents effectively compete with the water layer around the enzyme. Fluorescence spectroscopy shows that, although the presence of all the organic cosolvents cause conformational changes in the enzyme molecule at a concentration of 50% (v/v), these changes were completely reversible (when the concentration of organic solvent is diluted with aqueous buffer) in case of solvents having polarity indexes of 5.8 and above. In cases of the solvents having polarity indexes of 5.0 and below, the exposure at 50% concentration changed the conformation of the enzymes irreversibly. Thus, a simple parameter, viz. polarity index, may help in medium engineering of enzyme catalysis in nonaqueous surroundings.

A multipurpose immobilized biocatalyst with pectinase, xylanase and cellulase activities

BackgroundThe use of immobilized enzymes for catalyzing various biotransformations is now a widely used approach. In recent years, cross-linked enzyme aggregates (CLEAs) have emerged as a novel and versatile biocatalyst design. The present work deals with the preparation of a CLEA from a commercial preparation, Pectinex™ Ultra SP-L, which contains pectinase, xylanase and cellulase activities. The CLEA obtained could be used for any of the enzyme activities. The CLEA was characterized in terms of kinetic parameters, thermal stability and reusability in the context of all the three enzyme activities.ResultsComplete precipitation of the three enzyme activities was obtained with n-propanol. When resulting precipitates were subjected to cross-linking with 5 mM glutaraldehyde, the three activities initially present (pectinase, xylanase and cellulase) were completely retained after cross-linking. The Vmax/Km values were increased from 11, 75 and 16 to 14, 80 and 19 in case of pectinase, xylanase and cellulase activities respectively. The thermal stability was studied at 50°C, 60°C and 70°C for pectinase, xylanase and cellulase respectively. Half-lives were improved from 17, 22 and 32 minutes to 180, 82 and 91 minutes for pectinase, xylanase and cellulase respectively. All three of the enzymes in CLEA could be reused three times without any loss of activity.ConclusionA single multipurpose biocatalyst has been designed which can be used for carrying out three different and independent reactions; 1) hydrolysis of pectin, 2) hydrolysis of xylan and 3) hydrolysis of cellulose. The preparation is more stable at higher temperatures as compared to the free enzymes.

Methods in non-aqueous enzymology

1. Non-Aqueous Enzymology: Issues and Perspectives.- 2. Importance of Water Activity for Enzyme Catalysis in Non-Aqueous Organic Systems.- 3. Engineering of Enzymes via Immobilization and Post-Immobilization Techniques: Preparation of Enzyme Derivatives with Improved Stability in Organic Media.- 4 Immobilization of Lipases for Use in Non-Aqueous Reaction Systems.- 5. Applications of Enzymes and Membrane Technology in Fat and Oil Processing.- 6. Strategies for Improving the Lipase-Catalyzed Preparation of Chiral Compounds.- 7. Peptide Synthesis in Non-Aqueous Media.- 8. Enzyme Selectivity in Organic Media.- 9. Sugar Transformations Using Enzymes in Non-Aqueous Media.- 10. Reversed Micelles as Microreactors: N-Terminal Acylation of RNase A and its Characterization.- 11. Analysis in Non-Aqueous Milieu Using Thermistors.- 12. Importance of the Medium forin vitroandin vivoProtein Folding Mechanisms: Biomedical Implications.

The effect of ultrasonic pre-treatment on the catalytic activity of lipases in aqueous and non-aqueous media

BackgroundUltrasound has been used to accelerate the rates of numerous chemical reactions, however its effects on enzymatic reactions have been less extensively studied. While known to result in the acceleration of enzyme-catalysed reactions, ultrasonication has also been shown to induce enzyme inactivation. In this study we investigated the effects of ultrasonic pretreatment on lipases in both aqueous and non-aqueous media.ResultsOur results show that the ultrasonic pre-irradiation of lipases (from Burkholderia cepacia and Pseudomonas fluorescens) in aqueous buffer and organic solvents enhanced enzymic activities. In addition, we report the enhancement of hydrolytic (esterase) and transesterification activities.On using pre-irradiated enzyme, we found that the conversion rate for the transesterification of ethyl butyrate to butyl butyrate, increased from 66% to 82%. Similarly, a 79% conversion of Jatropha oil to biodiesel was observed upon employing pre-irradiated enzyme, in contrast to a 34% conversion with untreated enzyme.CD spectra showed that while the enzymes secondary structure remained largely unaffected, the microenvironments of aromatic amino acids were altered, with perturbation of the tertiary structure having also occurred. SEM analysis demonstrated significant morphological changes in the enzyme preparation as a result of ultrasonication.ConclusionIn contrast to the effects of ultrasonic irradiation on other enzymes, for the lipases focused upon in this study, we report an enhancement of biocatalytic activity, which is thought to originate from morphological changes on the macro and molecular levels.

Immobilized metal affinity chromatography without chelating ligands: purification of soybean trypsin inhibitor on zinc alginate beads.

Immobilized metal affinity chromatography (IMAC) is a widely used technique for bioseparation of proteins in general and recombinant proteins with polyhistidine fusion tags in particular. An expensive and critical step in this process is coupling of a chelating ligand to the chromatographic matrix. This chelating ligand coordinates metal ions such as Cu2+, Zn2+, and Ni2+, which in turn bind proteins. The toxicity of chemicals required for coupling and their slow release during the separation process are of considerable concern. This is an important issue in the context of purification of proteins/enzymes which are used in food processing or pharmaceutical purposes. In this work, a simpler IMAC design is described which should lead to a paradigm shift in the application of IMAC in separation. It is shown that zinc alginate beads (formed by chelating alginate with Zn2+ directly) can be used for IMAC. As “proof of concept”, soybean trypsin inhibitor was purified 18‐fold from its crude extract with 90% recovery of biological activity. The dynamic binding capacity of the packed bed was 3919 U mL‐1, as determined by frontal analysis. The media could be regenerated with 8 M urea and reused five times without any appreciable loss in its binding capacity.

Synthesis of Antioxidant Propyl Gallate Using Tannase from Aspergillus niger van Teighem in Nonaqueous Media

Tannase from Aspergillus niger van Teighem has been used for synthesis of food additive antioxidant propyl gallate by direct transesterification of tannic acid. The optimized yield of 86% was obtained by using simultaneously pH tuned enzyme, immobilized on Celite and using the right amount of water in the non aqueous media.

Enhancement of enzyme activity in aqueous-organic solvent mixtures

SummaryThe activities of poyphenol oxidase, peroxidase, acid phosphatase and trypsin were measured in the presence of four different water miscible solvents, acetonitrile; N,N dimethylformamide; tetrahydrofuran and dioxane. Stimulation of enzymatic activities was observed in all the cases at a specific concentration range of cosolvent added which cannot be correlated either with log P or denaturation capacity. Also, no significant structural changes were observed by fluorescence studies even at the cosolvent concentrations where activity increases were significant.

Affinity precipitation of proteins.

Affinity precipitation is being studied as a technique to be introduced at an early stage of downstream processing for the selective isolation of proteins. The technique utilizes a heterobifunctional ligand, which, in addition to having affinity for the target protein(s), possesses another function for controlling precipitation. The latter component is comprised of a polymer which can be made reversibly soluble and insoluble by altering a specific parameter such as pH or temperature. Different polymers of natural and synthetic origin have been used for this purpose. The soluble form of the ligand is used for the affinity binding step and precipitation is induced for obtaining separation of the affinity complex. Some of the polymers used in this laboratory include chitosan, alginate, Eudragit S‐100 (copolymer of methacrylic acid and methyl methacrylate) and polyethyleneimine. Chitosan and alginate served as natural ligands for wheat germ agglutinin and pectinase, respectively. The aromatic dye Cibacron Blue 3GA coupled to Eudragit S 100 and polyethyleneimine way used for the affinity precipitation of some model enzymes such as lactate dehydrogenase and alcohol dehydrogenase. As prior removal of cell debris, etc., is essential for affinity precipitation, the possibility of integration of the technique with extraction in aqueous two‐phase systems was also demonstrated.